PAS-CLASH (PolyA Site-Crosslinking, Ligation and Sequencing of the Hybrid) analysis to detect RNA-RNA interaction at poly(A) site region

In an attempt to discover potential RNA-RNA interactions in mRNA 3’ processing regulation, we developed PAS-CLASH (PolyA Site-Crosslinking, Ligation and Sequencing of the Hybrid) method, which combines reported CLASH and poly(A) site sequencing protocols. Briefly, HeLa cells were treated with AMT, or without AMT as control, and irradiated at 365 nm for crosslinking. Following nuclear RNA fragmentation and purification, the potential proximate RNAs/PASs were ligated on the oligo d(T)25 beads, hybrids were reverse crosslinked and amplified with the QuantSeq Rev 3' mRNA sequencing library prep kit (Lexogen, Cat. 016.24) and sequenced with Hiseq, producing 150 bp paired end reads. For each sample (AMT treatment or without treatment), we have 2 biological replicates, in total there are 4 samples. Potential RNA-RNA interactions is supposed to be enriched in AMT treated samples